The major objective of Project 2 is to use mechanistic enzymology and x-ray crystallography to understand the structure and function of two proteinases, SCLpro and PLpro, that are essential for the virulence of the SARS coronavirus. Structural and mechanistic information will be critical to the structure-based design efforts of the overall Program Project that has the goal of designing potent inhibitors of SCLpro and PLpro that will eventually be developed as therapeutic drugs. The specific aims of Project 2 are: (1) To determine the kinetic and chemical mechanisms of SARS SCLpro and PLpro as well as the substrate specificity of these enzymes using steady-state kinetics and site-directed mutagenesis approaches. (2) To determine the x-ray crystal structures of wild-type and mutant SARS SCLpro enzyme in complex with various substrates and inhibitors. (3) To crystallize and determine the x-ray structure of the SARS PLpro enzyme; and (4) develop novel high-throughput fluorescence screening (HTS) assays for SCLpro and PLpro proteinases that will allow for rapid screening of potential inhibitors from libraries of thousands of compounds. Once potent inhibitors are developed via collaboration with Project 3, we will then in collaboration with Project 1, use molecular biology approaches and x-ray crystallography in an attempt to identify the amino acids in the SARS SCLpro and PLP2 sequences that could give rise to drug resistance via mutation. We have already cloned and over-expressed active constructs of both SCLpro and PLpro, and we have crystallized and determined the x-ray structures of SCLpro in complex with three inhibitors, synthesized by Project 3, that inhibit SARS-CoV replication in vivo. We have developed a continuous fluorescence assay for SCLpro that will allow us to conduct our steady-state kinetic studies, and we have established a collaboration with an industrial partner, PharmOptima LLC, who will help us more fully develop and optimize our fluorescence assays so that we can rapidly screen large compound libraries. The results from our proposed experiments will provide important information to the Program Project that will be crucial for the development of novel inhibitors that may be used as therapeutic agents to reduce SARS-CoV replication and pathogenesis.